ECOLOGY and BIOMECHANICS - CHAPTER 6 doc

The forces exerted by visiting insects and the concomitant deformations of the flower cause a relativeforward movement of style and stigma in the staminal tube that squeezes a portion of

Flowers: The Role of Lever and Flower Tube in Specialization on

Pollinators

Martin Reith, Regine Claßen-Bockhoff, and Thomas Speck

CONTENTS

6.1 Introduction 124

6.1.1 Biomechanics and Bee Pollination 124

6.1.2 A Case Study: The Staminal Lever Mechanism in Salvia 125

6.2 Materials and Methods 126

6.2.1 Materials 126

6.2.2 Forces of Flower-Visiting Bees 127

6.2.3 Force Measurements on Salvia Flowers and Staminal Levers 132

6.3 Results 134

6.3.1 Forces Exerted by B terrestris and A mellifera 134

6.3.2 Forces and Flower Visitors of Salvia 134

6.4 Discussion 136

6.4.1 Insect Forces 136

6.4.2 Observed Flower Visitors 137

6.4.3 Forces Measured in Salvia Flowers 138

6.4.3.1 Critical Discussion of the Applied Methods 138

6.4.3.2 Comparison of Levers and Internal Barriers in Flowers 139

6.4.4 Comparing Insect Forces to the Barriers in Flowers 140

6.4.5 Proboscis Length, Flower-Tube Length, and Forces Exerted by Visiting Bees 140

6.5 Conclusion 141

Acknowledgments 143

References 143

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124 Ecology and Biomechanics

6.1 INTRODUCTION

6.1.1 B IOMECHANICS AND B EE P OLLINATION

Biomechanical interactions between bees and flowers have been known to be involved

in pollen transfer in plant species for more than two centuries [e.g., 1–6] Bees arevery important pollinators for many plant species However, because bees feed theiroffspring nearly exclusively with pollen, plants have evolved several mechanisms toavoid overexploitation by pollen-collecting bees and to ensure pollen transfer [7–11].Many of these mechanisms involve specific mechanical features Four of the mostimportant are buzz pollination and the piston, brush, and lever mechanisms

Buzz pollination is well-known in many economically important members of thenightshade family (Solanaceae) [12] Examples include tomato, Solanum lycopersicum;pepper, Capsicum annuum; and eggplant, S melongena However, buzz pollination ismuch more widespread and known to occur in at least 65 plant families [13] Thethecae of many of these plants dehisce only partially, and they only open small poresthrough which pollen can be released (poricidal anthers) To collect pollen from theseplants, bees place their body near the small openings and vibrate the stamen by rapidcontraction of their indirect flight muscles Buchmann and Hurley [13] model theprocess of pollen release The bee’s buzzing and the consequential vibration of theanther wall transmit energy to the pollen grains inside the anther Thus, the energycontent of the anther increases while pollen grains accumulate more and more kineticenergy by repeated interactions with the walls and with each other Release of pollen

on the other hand diminishes the energy content of the anther If buzzing continues,the number of pollen grains in the anther and the number of pollen grains that escapethe anther diminish, but their average kinetic energy increases Although this model

is an extreme simplification of flower morphology, it is a cornerstone in the standing of buzz pollination In the case of buzz pollination, overexploitation of thepollen is prevented because only a limited amount of pollen can be “buzzed out” during

under-a single visit It would be interesting to test if the size of buzzing pollinunder-ators under-and theenergy they can produce are related to morphological characters of the poricidal anthersand the energy needed to buzz the flowers of a given plant species, thereby limiting

“mechanically” the pollinator spectrum

The piston mechanism [14] often found in the pea family (Fabaceae) acts, from

a functional point of view, as a pollen pump The stamens that are hidden in thecarina have coalesced filaments and form a tube that encloses the style Flowervisitors have to deform the flower actively to reach the nectar The forces exerted

by visiting insects and the concomitant deformations of the flower cause a relativeforward movement of style and stigma in the staminal tube that squeezes a portion

of the pollen mass out of the staminal tube By this mechanism, pollen is transferred

to the ventral part of the pollinator’s body During a single visit, only a small amount

of pollen is extruded by the pollen pump, ensuring pollen dispensing

The brush mechanism, which also occurs in the pea family, works in a similarmanner [15] In Lathyrus latifolius, a stylar brush underneath the stigma takes thepollen up during flower development Later when the ripe flower is deformed byforces exerted by a visiting insect, the carina is lowered and the stigma and the stylar

Biomechanics of Salvia Flowers 125

brush touch the insect consecutively, thus avoiding self-pollination In flowers of

Lathyrus latifolius, 100 millinewtons (mN) are needed to trigger this mechanism[16] (measured with a spring balance) Pollen dispensing is realized by the brush,which deposits only a dosed amount of pollen on each visitor

Staminal levers in the broadest sense, i.e., stamina that can be tilted with orwithout a hinge, occur not only in the genus Salvia (see Section 6.1.2) but are alsofound in the Lamiaceae–Prostantheroideae [17,18] and in other families such as theZingiberaceae An example from the latter family is Roscoea purpurea [19] As theauthor points out, the structure of its stamen differs significantly from the Salvia

lever Flowers of R purpurea have only one fertile stamen; the two thecae areextended; and their basal parts are sterile and block the flower entrance The upperpart of the thecae is fertile and produces pollen Salvia, in contrast, has two fertilestamens and a different stamen morphology, as described in more detail below Inboth taxa, a flower visitor releases the mechanism by pushing against the lever arm,which extends into the flower tube Thereby, it is loaded with pollen on its head,neck, or back

6.1.2 A C ASE S TUDY : T HE S TAMINAL L EVER M ECHANISM INS ALVIA

The previous examples show that there exist many bee-pollinated plant species inwhich sophisticated mechanisms are involved in pollen presentation, pollen dispens-ing, and/or pollen transfer In the present paper, we focus on the staminal levermechanism found in sages (genus Salvia) Form, function, and ecology of flowers

of the genus Salvia have been recently reviewed [20,21] We therefore only marize briefly the basic morphological and biomechanical parameters of the Salvia

sum-flower and its staminal lever mechanism, which are important for our studies Flowersare sympetalous and mainly bilabiate with an upper and a lower lip (Figure 6.1).Four stamens are usually formed, the upper (adaxial) pair being reduced to small tominute staminodes and the lower (abaxial) pair forming the lever mechanism Ineach stamen, the lever arms are formed by the extended connective that is fixed tothe filament by a miniature joint-like ligament The lower lever arm is sterile inmany species, while the upper one is always fertile, producing one theca The twostamens are often partially fused, thus forming a functional unit Pollinators in a

“typical” Salvia flower push against the base of the sterile lever arm (BC in Figure6.1), causing a swing of the lever around the jointlike ligament (JL) so that thevisitor is touched by the pollen sacs located at the upper, fertile lever arm (UC) andbecomes loaded with pollen on its back or head In later flower development, stigmasare generally orientated to occupy the same position as the previously functionalpollen sacs A pollinator carrying pollen delivered from the staminal levers is there-fore likely to transfer the pollen to the stigma of a flower of the same species.Mechanical barriers that limit the access to floral food sources are one of themost important mechanisms that promote specialization in pollination systems[22,23] Since Salvia flowers provide nectar as a main food source to their pollinators,

a crucial question is if and how Salvia flowers restrict access to the nectar to alimited range of flower visitors Having the typical structure of a Salvia sympetalousflower with the staminal lever mechanism and nectar presentation at the base of a

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126 Ecology and Biomechanics

corolla tube in mind, there are two different mechanical exclusion mechanismspossible: (1) the length and diameter of the flower tube might be an important factor

in restricting access to the nectar [24,25, Claßen-Bockhoff and Kuschwitz (in aration)], and (2) flower visitors that are not able to move the staminal lever may

prep-be mechanically excluded from the nectar by this rigid barrier [21,26] The odological approaches we have developed allow for a quantitative measurement ofthe forces insects are able to exert while gaining access to an artificial nectar source.The methods we have developed allow us to measure (1) the forces that insects areable to exert while gaining access to a custom-made artificial nectar source (Figure6.3), and (2) the forces necessary for insects to trigger the staminal lever and toforce themselves along the corolla tube towards the nectar source (Figure 6.4) Thisenables us to test the different barrier hypotheses and to reconsider the functionalimportance of different flower structures for flower-pollinator specialization

meth-6.2 MATERIALS AND METHODS

6.2.1 M ATERIALS

We studied honeybees (Apis mellifera) and bumblebees (Bombus terrestris) as plary species Apis mellifera workers have a body length of 12.1 ± 0.3 mm (n = 10,mounted specimens), and a mean proboscis length between 6.05 and 6.40 mm in

exem-FIGURE 6.1 Schematic longitudinal section of a “typical” Salvia flower showing the tioning of the staminal lever mechanism For clarity, the style and second stamen are omitted Black: staminal lever arm in unreleased position, white: in released position UL: upper lip

func-of the flower, LL: lower lip func-of the flower, F: filament, JL: jointlike ligament, UC: upper part

of the elongated connective with fertile theca (FT), BC: basal, sterile part of the connective, N: nectar glands (Modified from Speck, T et al., in Deep Morphology: Toward a Renaissance

of Morphology in Plant Systematics, T Stuessey, T., F Hörandl, F., and Mayer, V., Eds., Koeltz, Königstein, 2003, p 241.)

FT

UC

UL

JL F

BC

N LL

Biomechanics of Salvia Flowers 127

the three most often cultured subspecies [27] The European bumblebee B terrestris

was used as an example of a large bee species Bumblebees vary greatly in size

because workers can be much smaller than queens Body length of the tested workers

was 12.0 ± 0.8 mm (n = 10, mounted specimens), but queens can reach a body

length of 19 mm or more Compared with other European bumblebee species, B.

terrestris has a rather short proboscis of 8 to 9 mm length [28] The bumblebee

colonies were bought from a commercial supplier (Re-natur GmbH, D-24601

Ruh-winkel, Germany) Forces of bumblebees were measured in the laboratory, and forces

of A mellifera were measured in a private bee yard near Freiburg, Germany

Flowers of the following Salvia species were studied: S amplexicaulis Lam.,

S forskahlii L., S glutinosa L., S cf microphylla Kunth, S nilotica Juss ex Jacq.,

S nubicola Wall ex Sweet, S phlomoides Asso, S pratensis L., S sclarea L.,

S transsylvanica (Schur ex Griseb.) Schur, S uliginosa Benth., S verbenaca L., and S

viridis L We took the flowers from plants that were cultivated in the Freiburg Botanical

Garden (Southwestern Germany) Before flowers were analyzed mechanically, internal

structure and morphology of each species was studied in a number of longitudinal

sections and cross sections The species we were especially interested in are:

Jupiter’s distaff, S glutinosa L., is a yellow-flowered species that occurs in

European and Asian mountain forests S glutinosa has a simple lever

morphology in the sense of Himmelbaur and Stibal [29] The connective

is strongly curved, and the flower entrance is not completely blocked by

the lever (Figure 6.2A and Figure 6.7A) Flower-visiting insects on S.

glutinosa were studied during the flowering periods in 2002 to 2004 in the

Freiburg Botanical Garden and in 2003 to 2004 on a further site near

Eichstetten (southwestern Germany) where this species grows naturally

Flowers are usually visited by bumblebees (see Table 6.1) and not by

honeybees or other midsized bees except for some pollen-thieving or

nectar-robbing visits

Clary sage, S sclarea L., is a well-known, light blue or pink to white flowering

Mediterranean species that is widely cultivated for its essential oils and as

an ornamental This species is a representative of the genus with a derived

staminal lever in the sense of Himmelbaur and Stibal [29] The lever is

spoon shaped and blocks the flower entrance completely (Figure 6.2B and

Figure 6.7B) In 2003 and 2004, we observed flower-visiting insects on S

sclarea in the Freiburg Botanical Garden and in private gardens in

Boetz-ingen (southwestern Germany) and Schwanau (southwestern Germany)

where this plant was cultivated Clary sage is visited mainly by carpenter

bees Xylocopa violacea and, as in S glutinosa, not by honeybees or other

midsized bees (Table 6.1)

6.2.2 F ORCES OF F LOWER -V ISITING B EES

Forces that insects are able to exert to gain access to a food source were measured

during visits to a custom-made “artificial flower” for which the insects can be trained

(Figure 6.3) The artificial flower consists of a base plate connected to a highly

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128 Ecology and Biomechanics

sensitive force transducer (Burster Präzisionsmeßtechnik GmbH, D-76593Gernsbach, Germany) and a retractable tube behind the posterior wall of the con-struction The retractable tube contains a sugar solution (apiinvert, Südzucker AG,D-97195 Ochsenfurt, diluted to 50%) as a food source The posterior wall is covered

by a foam pad with a central hole giving access to the tube with the sugar solution

As the bees are feeding, the thin tube containing the sugar solution is manuallyretracted By trying further to reach the food source, the insects start pressing theirheads against the foam pad The induced reactive force is transmitted via the bees’legs and the platform to the force transducer that measures this force with an accuracy

of ± 50 μN The force transducer converts the forces into a voltage output, which

is stored online by a laptop computer The tested bumblebees were continuously fedwith air-dried pollen, and the supply of the colony with sugar solution was interrupted

24 hr before the experiments were started

(A)

(B)

FIGURE 6.2 (A) S glutinosa flowers with B hortorum leaving a flower after a visit; original

habitat near Eichstetten (B) S sclarea visited by X violacea (private garden in Schwanau).

Flower Visitors of the Specialized S sclarea and S glutinosa, and the Less Specialized S pratensis

Ecology and Biomechanics

TABLE 6.1 (CONTINUED)

Flower Visitors of the Specialized S sclarea and S glutinosa, and the Less Specialized S pratensis

Source: Data was obtained either from the literature (21,32–34,41,50–57) or from this paper’s authors, represented by X Activity by the flower visitors was evaluated by the respective authors

or was based on descriptions from the references: + pollinator; 0 visitor (or no details given); N nectar robber; n nectar thief; p pollen thief; [ ] the original author used a synonym.

Copyright © 2006 Taylor & Francis Group, LLC

132 Ecology and Biomechanics

6.2.3 F ORCE M EASUREMENTS ON S ALVIA F LOWERS AND S TAMINAL

L EVERS

The forces a bee would have to exert during a visit on a Salvia flower were measured

with a custom-built force-measuring device (Figure 6.4) that mechanically mimicsthe visit of a pollinator [30] The very sensitive force transducer (Burster Präzision-smeßtechnik) and a displacement transducer (Burster Präzisionsmeßtechnik) are bothmounted on a micromanipulator that is driven by a low vibration DC-micromotorand a gearing system (Märzhäuser GmbH, D-35579 Wetzlar, Germany) The com-puterized instrument is powered by rechargeable batteries The measuring device ismounted on a portable tripod with a coordinate system setting that allows a straightpositioning of the force transducer and the force sensor to the flower The movements

of the sensor are controlled by a laptop computer that also stores the ment data This allows for a constant, predefined movement of the sensor into the

force–displace-flower and a measurement of forces even in fragile objects like Salvia force–displace-flowers with

an accuracy of ± 50 μm and ± 50 μN, respectively

FIGURE 6.3 Schematic drawing of the artificial flower, a custom-made device for measuring

forces exerted by insects to gain access to a food source (for description, see the text) (a) force transducer, (b) base plate, (c) sugar solution, (d) retractable food source, and (e) foam pad.

b

a c

d

e

Biomechanics of Salvia Flowers 133

For the initial tests on different Salvia species, the sensor was a thin metal rod

with a diameter of 0.60 mm This should mimic a bee’s proboscis (proboscis width

in A mellifera = 0.61 ± 0.05 mm, n = 10) In a first insertion cycle, we controlled

for correct positioning of sensor and flower Furthermore, we determined the internal,biologically sensitive flower-tube length, as indicated by a strong increase of themeasured forces when reaching the flower bottom When further measurements onthe same flower were conducted, the cycling distance was chosen according to theinternal flower-tube length For these measurements of forces necessary to releasethe staminal lever, the flowers were fixed in their original orientation on their pedicel

To test whether honeybees might be excluded from the nectar in S sclarea by

the dimensions of the corolla tube, we measured the forces that bees with a definedproboscis length have to exert to reach the nectar For this purpose, we used amodified sensor (Figure 6.5) A honeybee’s head was mounted on the thin metalrod We used a sliding caliper to adjust the distance between the sensor’s tip andthe bee’s head (edge of clypeus) to match the proboscis length that should besimulated Before each measurement, the bee’s head and the sensor were wettedwith silicone oil to reduce friction and sticking due to the lack of head motility.During the first insertion cycle, a sufficient proboscis length was chosen for thesensor tip so as to avoid contact of the bee’s head with the flower tube; this allowed

us to determine the internal flower-tube length In subsequent measurements on thesame flower, this internal flower-tube length was used as a maximum cycling dis-tance For this type of measurements, the flowers were fixed on their lower lip, whichreflects the natural situation in which bees typically use the lateral lobes of the lowerlip as grips when collecting pollen and nectar This type of fixation allows forrepeated measurements as the flowers show only very small spatial movements evenunder the repeated exertion of (comparatively) high forces

FIGURE 6.4 Schematic drawing of the “pollination simulator,” a custom-made device for

measuring forces necessary to release the staminal levers and to get access to the nectar gland

of Salvia flowers (for description, see the text) (a) Micromanipulator with distance transducer,

(b) force transducer, (c) sensor, and (d) low vibration motor.

c

d

b

a